The objectives of this proposal are to identify the structural features of lipoprotein lipase (LPL) required for binding to GPIHBP1 and to define mechanisms by which LPL is transported across endothelial cells. We discovered that GPIHBP1, an endothelial cell protein, binds LPL and is absolutely required for the lipolytic processing of triglyceride-rich lipoproteins. We showed that an absence of GPIHBP1 in mice causes severe hypertriglyceridemia (chylomicronemia) and that humans with GPIHBP1 mutations exhibit the same phenotype. Initially, we proposed that GPIHBP1 was a binding site for LPL in capillaries, but we recently found that GPIHBP1 has a second role-transporting LPL from the extracellular spaces into the capillary lumen. The identification of GPIHBP1 as the LPL transporter solved a longstanding mystery of plasma lipid metabolism, posing many new questions for investigation. Among these are: What amino acid sequences within LPL are important for binding to GPIHBP1? Do certain LPL mutations cause disease by abolishing LPL's ability to bind to GPIHBP1? Do other mutations enhance LPL transport? What is the efficiency of LPL transport across endothelial cells? Is LPL movement across capillaries bidirectional? Is LPL transported across capillaries in transcytotic vesicles? We have begun to investigate each of these issues. We have identified amino acid substitutions within the carboxyl terminus of LPL that abolish its ability to bind to GPIHBP1. These mutations have provided important clues regarding the location of LPL's GPIHBP1-binding domain, but many more studies are required to fully understand LPL-GPIHBP1 interactions. We have made great progress in understanding mechanisms of LPL transport, both in vitro and in vivo. Our studies have taken advantage of a host of new antibody reagents and expertise with both confocal and electron microscopy. We have two specific aims, each designed to better understand lipolysis in health and disease. In Aim 1, we will test the hypothesis that the carboxyl terminus of LPL is crucial for binding GPIHBP1 and examine LPL mutations that interfere with GPIHBP1 binding and transport across endothelial cells. In Aim 2, we will test the hypothesis that LPL is transported across endothelial cells by caveolar-mediated transcytosis. We are excited by these aims, and are poised-with all of the necessary expertise, reagents, and experimental techniques-to carry out the proposed studies.